Development of the gut in Xenopus laevis

The lining of the gut, together with the pancreas, liver, gall bladder, and respiratory system, is formed from the endoderm. The gut also contains smooth muscle and connective tissue of mesodermal origin. The amphibian Xenopus laevis is potentially an excellent model organism for studying how the cells of the endoderm and mesoderm become programmed to produce these internal organs. However, the anatomical complexity of the coiled gut presents a problem in studying its development. In order to overcome this problem we here present a comprehensive guide to the anatomy and histology of the developing Xenopus gut. We use a simple dissection to display its anatomy and the expression of four endodermal markers (alkaline phosphatase, IFABP, XlHbox8, and endodermin). We present schematic diagrams that show how the gut is arranged in three dimensions and how this organisation changes during development. We also present drawings of histological sections of the gut which allow any region to be identified and so represent an atlas for working with sections. Finally, we describe the histology of the cells of the various organs of the gut. This histological identification may be necessary for the identification of parts following experiments in which the normal pattern is disturbed.     

Development of the interphotoreceptor matrix in Xenopus laevis

Xenopus laevis interphotoreceptor matrix (IPM) contains a relatively aqueous insoluble wheat germ agglutinin (WGA)-binding component containing unidentified sialoglycoconjugates (Wood et al [1984] J. Comp. Neurol. 228:299-307). The appearance of WGA-binding macromolecules in the IPM was assessed during late embryonic stages (32-45) and in retinal rudiment cultures, using lectin cytochemistry and Western blotting techniques. Metabolic labeling of the neural retina versus retinal pigment epithelium (RPE)-choroid of juvenile Xenopus with 35S-MET was also evaluated in vivo and in vitro. Lectin cytochemistry of eyes from developmental stages 32-42 demonstrated distinct WGA-ferritin-binding sites on the developing outer segment membranes and in the IPM compartment. At stages 44-46 extensive WGA-binding domains were present as an extracellular network with other randomly scattered domains near the retinal pigment epithelium. Retinal rudiments from stage 32-33 were isolated and allowed to differentiate in hanging drop culture (Hollyfield and Witkowsky [1974] J. Exp. Zool. 189:357-377) with or without an investing pigment epithelium. Cultures developing with RPE exhibited an elaborate IPM with an anastomosing meshwork of WGA-ferritin binding sites. In the absence of RPE only limited amounts of binding restricted to the immediate vicinity of the developing photoreceptor outer segment membranes was observed. When Western blots were probed with WGA-HRP, stage 32-45 retinas demonstrated a major WGA-binding band of 126 kD. Similar amounts of WGA-binding macromolecules were synthesized in preparations cultured in the presence or absence of the investing RPE. During development the major WGA-binding component is a 126-kD protein. Equivalent synthesis of this protein in the presence and absence of RPE suggests that the PE is not required for synthesis of this 126-kD component. These results suggest that the retina is the primary site of synthesis of the WGA-binding components of the Xenopus IPM, whereas the PE plays a principal role in their assembly and organization.     

Shh expression in developing and regenerating limb buds of Xenopus laevis

Analysis of the kinetic behaviour of a two-enzyme-system carrying out two consecutive reactions was investigated in macroheterogeneous biphasic media (octane/buffer pH 9.6, v/v = 1:1). The lipase-catalysed hydrolysis of trilinolein and the subsequent lipoxygenation of the liberated linoleic acid, were coupled in a modified Lewis cell with a well-defined liquid/liquid interfacial area. Trilinolein was dissolved in the organic phase and hydrolysed in the presence of Mucor javanicus lipase at the organic/aqueous interface. Linoleic acid, liberated after hydrolysis was transferred to the aqueous phase and reacted with lipoxygenase. This reaction consumed linoleic acid and produced hydroperoxides, which favoured the transfer of residual linoleic acid, since they possess surface active properties. Catalysis and transfer influenced each other reciprocally. At low substrate concentrations, cooperativity phenomena were observed in the experimental and also the modelled two-enzyme systems. When the initial substrate concentration was high, the kinetic behaviour of the two-enzyme system in a compartmentalised medium, seemed to be independent of the substrate concentration, unlike that observed in homogeneous monophasic enzymology. The numerical integration program used to model the two-enzyme system was based on results obtained in separate studies of the following three phenomena: (1) trilinolein hydrolysis in biphasic medium. (2) linoleic acid transfer across a liquid/liquid interface and (3) lipoxygenation in an aqueous media. Results obtained by modelling were similar to the results observed experimentally.     

Observations on the migration and proliferation of gonocytes in Xenopus laevis

The process of primordial germ cell formation in the normal course of development of Xenopus laevis was examined with a light microscope on paraffin and Epon sections of embryos or tadpoles, extending over the period from the gastrula to the feeding tadpole stage. Positional changes of gonocytes with development were nearly the same as those reported on the same species by Blackler (1958) and Whitington & Dixon (1975). The following points were newly demonstrated. Gonocytes which have been located in a deep endodermal position till mid tail-bud stage come to be located in a rather peripheral region of the endoderm cell mass at stage 31 (late tail-bud), suggesting that the initial step of migration of the gonocytes towards the future genital ridge has already begun at this stage. Gonocytes at stages 33/34 and 35/36 were observed in a more dorsal part of the endoderm than at stage 31. Gonocytes which seem to have begun their migration are roundish in external shape and have a large intercellular space around them. At stage 40 gonocytes were located in the dorsal endodermal crest, and at stage 41 gonocytes were found with cell bodies extending over both the dorsal endoderm crest and the dorsal mesentery, indicating that the separation of the gonocytes from the endoderm was in progress at this stage. The present results seem to indicate that gonocytes migrate not passively but actively from the deep endodermal position to the genital ridge, passing through the dorsal mesentery. Counting the number of gonocytes at successive stages of development revealed that gonocytes proliferated exponentially throughout the developmental stages from gastrula to tadpole.     

Identification of a multixenobiotic resistance mechanism in Xenopus laevis embryos

In the '90's a membrane-associated transport protein, discovered in aquatic organisms, was considered to be expressed in response to environmental xenobiotics. Like the multidrug resistance protein found in mammalian tumor cell lines, this protein confers resistance in organisms in polluted areas by binding xenobiotics and transporting them out of the cells in an energy-dependent manner. This study investigates the expression and the activity of a P-glycoprotein (Pgp) involved in a multixenobiotic resistance mechanism (MXRM) during the early developmental stages and in tissues of adult Xenopus laevis.     

Purification and some properties of ribonuclease from Xenopus laevis eggs

A 122 kDa RNase from eggs of Xenopus laevis was purified by sequential chromatography on Sephadex G-75, DEAE-cellulose, heparin-Sepharose and TSK gel G3000SW columns, and gave a single 60 kDa band on SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The RNase composed of two 60 kDa subunits is able to recognize pyrimidine bases specifically. The pH optimum of the RNase was 7.5 in Tris-HCl buffer. The enzyme activity was abolished by treatment at 80 degrees C for 5 min and pH 2 or 12 for 1 h. Since egg lectins with RNase activity obtained from Rana catesbeiana and R. japonica and bovine pancreatic RNase A show about 30% protein homology and these three proteins are 12-14 kDa heat-stable RNases, [K. Titani, K. Takio, M. Kuwada, K. Nitta, F. Sakakibara, H. Kawauchi, G. Takayanagi and S. Hakomori, Biochemistry, 26, 2189 (1987); Y: Kamiya, F. Oyama, R. Oyama, F. Sakakibara, K. Nitta, H. Kawauchi, Y. Takayanagi and K. Titani, J. Biochem. (Tokyo), 108, 139 (1990)], the data suggest that the X. laevis egg RNase is a unique protein compared with RNases from not only amphibians, but also mammals.     

The deleterious effects of fungicides and herbicides on Xenopus laevis embryos

Groups of 30 Xenopus laevis embryos, at "tail-bud" stage (Nieukoop-Faber stages 22-24) were exposed to 0.1-2 ppm concentrations of various pesticides for 1 to 10 days. The pesticides used were chloranil and dichlone (both are fungicidal and herbicidal); diquat (herbicide); and nabam (fungicide). The parameters examined were mortality, gross morphology, histology, and behavior. Chloranil (1.25 to 1.75 ppm) treated embryos showed abnormalities of the otolith, optic cup, and general pigmentation. Their movement was sporadically convulsive and they were unable to maintain proper balance. Dichlone (0.1 to 0.15 ppm) disrupted the development of the cephalic end of the embryo. Many of these embryos developed a slightly retarded trunk and tail only. These headless embryos lived for a time and were relatively lethargic. Diquat (0.75 to 2.0 ppm) administration reduced body size and pigmentation, and altered body shape. When embryos were treated with both 1.0 ppm of diquat and 2.0 ppm of nabam the integrity of myomeres and myocommata of the musculature was disrupted. The histological bases of these morphological and behavioral changes are discussed.     

On the existence of polyadenylated histone mRNA in Xenopus laevis oocytes

In a variety of systems, histone mRNA has been shown to lack poly(A) (Adesnik and Darnell, 1972; Grunstein et al., 1973). We have found, however, that in Xenopus laevis oocytes, poly (A)-containing mRNA codes for histones, in a wheat germ cell-free system, based on the following criteria: first, co-migration with authentic X. laevis oocyte histones on polyacrylamide gels; second, no detectable incorporation of tryptophan; third, differential incorporation of lysine and methionine into histone fraction H2A; fourth, resistance of histone fraction H2A to cleavage with cyanogen bromide; and fifth, correspondence of tryptic peptide maps of partially purified cell-free products with authentic X. laevis oocyte histone. RNA which directs the synthesis of histones in the cell-free system is retained on oligo(dT)-cellulose, even after denaturation in 80% DMSO at 70 degrees C, thereby demonstrating the covalent attachment of polyadenylic acid sequences to the mRNA. Poly (A)- RNA (7S-14S fraction) was also found to code for histones using the same criteria. We discuss the significance of the finding that X. laevis oocytes contain two classes of histone mRNA as well as the potential developmental implications of this observation.     

Neural crest formation in Xenopus laevis: mechanisms of Xslug induction

A study of the induction of the prospective neural crest in Xenopus laevis embryos has been carried out, using the expression of Xslug as a specific marker for the neural crest. We have analyzed the competence and the specification of the neural crest. The competence to express Xslug was analyzed using two different approaches: (1) in vitro culture of conjugates of dorsal mesoderm and ectoderm taken from embryos at different ages and (2) grafts of equivalent pieces of ectoderm in the neural fold region of a gastrula or a neurula. Similar results were obtained with both methods: the ectoderm loses the competence to respond to neural fold induction at the end of gastrulation. Neural crest specification was analyzed by culturing a region of the ectoderm that contained the prospective neural crest and analyzing Xslug expression. Our results show that neural folds are specified autonomously to express Xslug by the end of gastrulation. By grafting labeled neural plate into lateral epidermis we have shown that neural crest can be induced by an interaction between neural plate and epidermis. Furthermore, neural crest cells come from both tissues. We have discarded the possibility that these neural crest cells are induced by a signal coming from the underlying lateral plate, by a homeogenetic signal coming from the host neural plate, or by regeneration of crest cells from the dissected neural plate. We propose a model to explain how the neural crest cells are induced at the border of the neural plate in X. laevis.     

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in early Xenopus embryos induces cell dissociation and inhibits transition from the blastula to gastrula stage

Xenopus early embryos contain relatively low levels of S-adenosyl-methionine decarboxylase (SAMDC) and its mRNA. When SAMDC mRNA was injected into Xenopus embryos, it was preserved until the blastula stage and induced a large increase in SAMDC activity. The SAMDC-overexpressed embryos developed normally until the blastula stage but at the early gastrula stage cells which received the mRNA, dissociated autonomously and stopped synthesizing protein. In a hypotonic medium, the dissociated cells, and hence whole embryos, autolyzed. However, in isotonic media dissociated cells did not autolyze, although they did not divide and their DNA and RNA synthesis activity was greatly inhibited. The effects of SAMDC overexpression were abolished by coinjection of ethylglyoxal-bis(guanylhydrazone) (EGBG), a specific inhibitor of SAMDC. In SAMDC-overexpressed embryos the level of putrescine decreased and that of spermidine increased, though to limited extents, resulting in a considerable decrease in the putrescine/spermidine ratio. However, direct injection of spermidine did not mimic the effect of SAMDC overexpression, and putrescine coinjected with SAMDC mRNA to maintain the normal putrescine/spermidine ratio did not rescue the embryos. Conversely, the level of S-adenosylmethionine (SAM) greatly decreased and coinjection of SAM, which restored the level of SAM, rescued the embryos. We concluded that in SAMDC-overexpressed embryos a SAM-deficient state was induced and this caused cell dissociation and inhibition of transition from the blastula to gastrula stage. We suggest that the SAM-deficient embryos obtained in the present study provide a unique system for studying the cellular control mechanism underlying the blastula-gastrula transition.     

Structural changes in oocyte nucleoli of Xenopus laevis during oogenesis and meiotic maturation

Immunoelectron microscopy with anti-nucleolin defined substructures within the multiple nucleoli of biosynthetically active stage II-III oocytes and within the nucleoli of relatively quiescent stage VI oocytes of Xenopus laevis. Dense fibrillar components (DFCs) of nucleoli from stage II-III oocytes consisted of nucleolonemas that radiated from a continuous DFC sheath surrounding fibrillar centers (FCs). Discernible granular regions (GRs) were absent in these same nucleoli. Conversely, stage VI oocyte nucleoli displayed compacted DFCs and prominent GRs. Immunofluorescence microscopy then tracked fibrillarin, nucleolin, and condensed DNA through oogenesis and into progesterone-induced meiotic maturation and nuclear breakdown. In stage II-III oocyte nucleoli, fibrillarin was enriched near the FC-DFC boundaries, while nucleolin was distributed throughout these same DFCs. Both proteins were enriched within the compacted DFCs of stage VI oocyte nucleoli. Staining with (DAPI) 4',6-diamidino-2-phenylindole showed condensed DNA within nucleolar FCs of both stage II-III and stage VI oocyte. Upon nuclear breakdown, we found fibrillarin and nucleolin in small particles and in the surrounding cytoplasm. Although we saw no trace of fibrillarin or nucleolin in nuclear remnants prepared just minutes later, DAPI-stained particles remained within these preparations, thus suggesting that FCs were at least slow to disassemble.     

Identification of a novel member of the pentraxin family in Xenopus laevis

Pentraxins are a family of acute phase reactants. Two family members, C-reactive protein (CRP) and serum amyloid P component (SAP), are known in a range of mammalian species. CRP and SAP are both about 200 residues long, and arose from a gene duplication event, apparently before the divergence of the mammalian orders. To elucidate the origins of mammalian pentraxins, we have searched for pentraxin-coding genes in the amphibian Xenopus laevis. We have identified a gene determining a protein (XL-PXN1) which is about twice the size expected: the XL-PXN1 gene appears to be a fusion between regions encoding an amino-terminal peptide of unknown function and a carboxy-terminal pentraxin. The pentraxin domain is more divergent from CRP and SAP than they are from each other: it provides an outgroup for analysis of the evolution of mammalian pentraxins and confirms that putative CRP and SAP proteins partly characterized in non-vertebrate species cannot be true homologues of the mammalian proteins.     

Solubilization and biochemical characterization of the melatonin deacetylase from Xenopus laevis retina

Melatonin deacetylase, an enzyme activity recently discovered in the Xenopus laevis retina, regulates local melatonin levels. The deacetylase occurs in retina, retinal pigment epithelium, and skin, all sites of melatonin action, and is widely distributed among vertebrates. We have solubilized the enzyme from Xenopus retina and pigment epithelium using nonionic detergents, and have developed a specific enzyme assay. We have characterized the enzyme and now report that the deacetylase is relatively specific for melatonin and is inhibited by the melatonin precursor N-acetylserotonin and the product of the deacetylase, 5-methoxytryptamine. Inhibition of deacetylase activity by eserine (physostigmine) suggests a relationship between deacetylase and cholinesterase activities. However, among a variety of cholinesterase inhibitors tested, only eserine inhibits the deacetylase. Furthermore, eserine is much less potent as an inhibitor of the deacetylase than the cholinesterases, and purified cholinesterases failed to deacetylate melatonin. We also show that melatonin deacetylase and aryl acylamidase (an enzyme related to cholinesterases) activities are differentially extractable from Xenopus ocular tissues, and that they exhibit different pH optima and inhibition profiles. Our results provide an initial characterization of the Xenopus retinal melatonin deacetylase, and indicate that deacetylase activity is distinct from cholinesterase and aryl acylamidase activities.